The Rb tumor suppressor (pRb) plays a critical role in stress erythropoiesis. We have shown that under stress conditions, such as hemolytic anemia, bone marrow transplant or tumorigenesis, pRb is required to regulate erythroblast expansion and to coordinate cell cycle exit with enucleation. Loss of pRb resulted in aplastic anemia and depletion of stem cells and progenitors from bone marrow and spleen. However, the underlying mechanisms that explain the critical role of pRb in stress erythropoiesis are not known. We hypothesize that the Rb tumor suppressor regulates a differentiation checkpoint in erythroblasts that is sensitive to oxidative stress and levels of DNA damage. We shall determine whether oxidative stress and/or DNA damage affects the ability of erythroblasts to exit cell cycle, differentiate and mature by enucleating and whether the ability to do so is dependent on functional pRb (Aim 1). Furthermore, we shall characterize the effects of Rb loss on expression of key modulators of DNA repair and oxidative stress, including red cell antioxidants. We also propose that E2f-2 is the key E2f target of pRb in post-mitotic erythroblasts and that by understanding how E2f-2 is regulated and by identifying physiologically relevant target genes, we shall understand why pRb is critical for stress erythropoiesis. We shall identify the upstream signaling pathways that promote expression of E2f-2 and are required to induce growth arrest of erythroblasts (Aim 2). We shall characterize how these signaling pathways impinge upon transcriptional regulation of E2f-2 by identifying the transcription factors that bind to and activate the E2f-2 promoter. Finally, we shall identify and validate genes that are regulated by E2f-2 and/or pRb in differentiating erythroblasts that explain aspects of the role played by pRb/E2f-2 in modulating oxidative stress, DNA damage and maturation of red cells (Aim 3). Thus by examining how the ability of erythroblasts to manage oxidative stress, repair DNA damage and undergo checkpoint arrest affects their differentiation potential, and how this in turn is regulated by pRb and E2f-2, we shall shed light on how the proliferative response to anemia is attenuated following anemic stress and how aplastic anemia, myelofibrosis and other blood disorders develop in humans.